The invention relates to a new use of prothrombin fragments, in particular, of meizothrombin, meizothrombin (desF1), or mixtures thereof.
The determination of thrombin substrates by activation by means of thrombin constitutes an important methodology of characterizing blood and plasma products. Thus, concentrations or activities of fibrinogen, factor V, factor VIII or protein C, for instance, are normally determined by activation by means of thrombin in plasma-containing samples.
Thrombin is a 36 kD protein that plays a leading role in the blood coagulation cascade, its main function being the transformation of fibrinogen to fibrin, the latter forming a fibrin network by aid of factor XIII also activated by thrombin.
Moreover, thrombin also activates blood coagulation factors, such as factor V, factor VIII and protein C, causing the aggregation of thrombocytes. A number of other proteins, such as fibronectin, thrombospondin and apolipoprotein, various collagens, aminin, etc. (cf. Table 1 in Stocker, Sem. Thr. Hem. 17 (2) p. 114 (1991)) likewise can be cleaved by thrombin. In the following description, such proteins are denoted as "thrombin substrates".
On account of its important position in the coagulation cascade, the action of thrombin in vivo is very precisely controlled by activity effectors or inhibitors.
However, in particular, these inhibitors interfere with the assaying of thrombin substrates, because the inhibitors falsify the results in a sample, or a much larger, excessive amount of thrombin must be added prior to assaying in order to outplay the inhibitors present.
An important thrombin inhibitor, for instance, is heparin in combination with antithrombin III. Heparin practically is contained in any blood sample, either if blood donations are heparinized, i.e., admixed with heparin, immediately after having been taken to prevent premature coagulation or if the patient has obtained heparin, e.g., in an anticoagulation therapy.
The thrombin-inhibiting agent proper is antithrombin III. Heparin potentiates the effect of antithrombin III.
In a heparin-containing blood or plasma sample, there is always the risk of getting falsified results in thrombin concentration, thrombin substrate activation or kinetic assays, unless it has previously been freed from heparin in a cumbersome manner or heparin has been neutralized.
This involves an additional process step, which is hardly advantageous in diagnostic methods to be feasible in as simple a manner as possible for purposes of routine. Any additional process step not only involves higher operational expenditures, but also constitutes a potential additional error source of the assaying process.
Thrombin is formed in blood from an inactivated precursor protein, i.e., prothrombin, to a mature enzyme via several intermediate steps. The mature enzyme consists of two chains (A and B chains) interconnected by a disulfide bridge.
Meizothrombin or meizothrombin (desF1) are prothrombin fragments of this type, having been formed by the incomplete activation of prothrombin to thrombin (cf. FIG. 1 in Doyle et al., J. Biol. Chem. 265 (18), 10693-10701 (1990)). Meizothrombin (desF1) is formed by the activation of prothrombin in the absence of a thrombin inhibitor. In doing so, meizothrombin is first formed, which is proteolytically degraded to meizothrombin (desF1) after cleavage of the fragment F1. Thus, the molecular weight is reduced from approximately 72 kD to approximately 48 kD. These fragments are said to be instable, because they degrade autocatalytically, meizothrombin, for instance, being rapidly transformed into .alpha.-thrombin by autocatalysis. Meizothrombin and meizothrombin (desF1) also may be produced by the action of the snake venom ecarin on thrombin.
Experiments carried out by Doyle et al. have demonstrated that, in contrast to prothrombin, bovine meizothrombin has a protease activity that is very low as compared to that of thrombin.
Although meizothrombin is capable of cleaving, e.g., fibrinogen into fibrin, or of activating protein C at a low rate, it is not inhibited by heparin, or the inibition effect is smaller by orders of magnitude (Stocker & Muller, Thrombosis and Haemostasis 65 (6), Abstract 855 (1991). However, it has also been shown that human prothrombin fragments are substantially more instable than, for instance, bovine ones.
But it is also known that some snake venoms cleave thrombin substrates. Andersson (Haemostasis 1, 31-43 (1972)) describes a thrombin-like activity of several snake venoms, expressing the same in NIH units/ml analogous to thrombin. However, the use of snake venoms within the scope of diagnostics by human proteins has the disadvantage of inspecific interactions (intraspecies interactions). Therefore, efforts have been made to use only mammal proteins, preferably human proteins, particularly in the development of diagnostic methods for the determination of human factors. An essential prerequisite for an assaying method is the unambiguous reaction of the proteins used.